73 research outputs found
Pro-inflammatory cytokines affect pancreatic carcinoma cell. Endothelial cell interactions
OBJECTIVES: The potential role of surgery-induced pro-inflammatory\n cytokines on the development of tumor recurrence in pancreatic cancer was\n investigated. MAIN OUTCOME MEASURES: The adhesion of 3 human pancreatic\n carcinoma cell lines, PanC1, MiaPaCa and BxPC3 to monolayers of\n microvascular endothelial cells after pre-incubation with 0.1 or 10 ng/mL\n IL-1beta, TNF-alpha or IL-6 was assessed in a reproducible human in vitro\n assay. Untreated monolayers served as controls. RESULTS: Pre-incubation of\n microvascular endothelial cells with IL-1beta or TNF-alpha, but not IL-6,\n increased adhesion of all three tumor cell lines as compared to adhesion\n in the control group. Maximally stimulated adhesion for PanC1 reached\n 159%, for MiaPaCa 204% and for BxPC3 155% (all vs. the control, P<0.001).\n Pre-incubation of microvascular endothelial cells with IL-1beta or\n TNF-alpha resulted in a significant up-regulation of E-selectin, ICAM-1\n and VCAM-1 expression. The addition of anti-E-selectin, anti-ICAM-1 or\n anti-VCAM-1 monoclonal antibodies did not decrease adhesion to\n microvascular endothelial cells pre-incubated with IL-1beta. Therefore,\n enhanced tumor cell binding seems to be independent of these adhesion\n molecules. CONCLUSIONS: Pro-inflammatory cytokines derived from surgical\n trauma may enhance tumor cell adhesion to microvascular endothelial cells\n and thus bring about more successful tumor cell implantation resulting in\n an increased risk of metastasis formation
Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide
Recently, we developed a technique that allows the in vivo visualization
in man of somatostatin receptor-positive neuroendocrine tumors after i.v.
injection of [125I-Tyr3]octreotide or [111In-DTPA-D-Phe1]octreotide.
Radiotherapy of such tumors using somatostatin analogs coupled to alpha-
or beta-emitting radionuclides has been proposed as an application for
radiolabeled somatostatin analogs. To develop this concept further, it is
of importance to know whether the above-mentioned radiolabeled
somatostatin analogs are internalized by the tumor cells, and whether it
might be possible to manipulate the degree of internalization. In the
present study we investigated the internalization of a stable somatostatin
analog, [125I-Tyr3]octreotide, by mouse AtT20/D16V pituitary tumor cells
and primary cultures of human GH-secreting pituitary tumor cells.
Treatment of the cells with low pH was used to distinguish between
membrane-bound (acid-releasable) and internalize (acid-resistant)
radioligand. [125I-Tyr3]octreotide showed a time-dependent increasing
accumulation in AtT20 cells; after 4 h of incubation, values up to 6-8% of
the dose of radioligand added were obtained. Binding and internalization
of [125I-Tyr3]octreotide were temperature dependent and inhibited by
pertussis toxin. Inhibitors of lysosomal degradation did not increase the
amount of internalized radioligand. After 4 h of incubation, 88% of the
radioactivity present in the cells was still peptide bound, suggesting a
low intracellular breakdown of this radioligand. Six of seven human
GH-secreting adenoma cell cultures also internalized [125I-Tyr3]octreotide
(variation between 0.24-4.98% of the dose radioligand added). Displacement
of binding and internalization of [125I-Tyr3]octreotide by unlabeled
octreotide showed a bell-shaped curve in AtT20 cells. At low
concentrations (0.1 and 1 nM), binding and internalization were increased,
whereas at higher concentrations, saturation occurred. In contrast to
this, binding of [125I-Tyr3]octreotide to a broken cell preparation of
AtT20 cells was displaced in a dose-dependent manner by unlabeled
octreotide, with an IC50 of 0.1 nM. Similar observations were made in the
human GH-secreting adenoma cell cultures. In conclusion, a high amount of
[125I-Tyr3]octreotide is internalized in a specific-, time-, temperature-,
and pertussis toxin-sensitive GTP-binding protein-dependent manner by
mouse AtT20 and human GH-secreting pituitary tumor cells. In the presence
of a low concentration of unlabeled octreotide, a rapid increase in the
amount of [125I-Tyr3]octreotide internalized by AtT20 cells and by the
majority of the human GH-secreting adenoma cell cultures was
found.(ABSTRACT TRUNCATED AT 400 WORDS
Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas
Interferon-alpha (IFN alpha) may exert direct inhibitory effects on cell
proliferation and on the production of different peptide hormones. We
investigated the effect of IFN alpha on hormone production by 15
GH-secreting pituitary adenomas, 4 clinically nonfunctioning or
gonadotroph pituitary adenomas, and 4 prolactinomas in vitro. In the
GH-secreting pituitary adenoma cultures, a short term (72-h) incubation
with IFN alpha (50-100 U/mL) significantly inhibited GH secretion in 3 of
7 cases and PRL secretion in 6 of 7 cultures. During prolonged incubation
(14 days) with IFN alpha, GH and/or PRL secretion was significantly
inhibited in 7 of 8 cultures (GH, 17-78% inhibition; PRL, 39-88%
inhibition). In the clinically nonfunctioning or gonadotroph cultures
Applying HDACis to increase SSTR2 expression and radiolabeled DOTA-TATE uptake:from cells to mice
Aims: The aim of our study was to determine the effect of histone deacetylase (HDAC) inhibitors (HDACis) on somatostatin type-2 receptor (SSTR2) expression and [111In]In-/[177Lu]Lu-DOTA-TATE uptake in vitro and in vivo. Materials and methods: The human cell lines NCI-H69 (small-cell lung carcinoma) and BON-1 (pancreatic neuroendocrine tumor) were treated with HDACis (i.e. entinostat, mocetinostat (MOC), LMK-235, CI-994 or panobinostat (PAN)), and SSTR2 mRNA expression levels and [111In]In-DOTA-TATE uptake were measured. Furthermore, vehicle- and HDACi-treated NCI-H69 and BON-1 tumor-bearing mice were injected with radiolabeled DOTA-TATE followed by biodistribution studies. Additionally, SSTR2 and HDAC mRNA expression of xenografts, and of NCI-H69, BON-1, NCI-H727 (human pulmonary carcinoid) and GOT1 (human midgut neuroendocrine tumor) cells were determined. Key findings: HDACi treatment resulted in the desired effects in vitro. However, no significant increase in tumoral DOTA-TATE uptake was observed after HDACi treatment in NCI-H69 tumor-bearing animals, whereas tumoral SSTR2 mRNA and/or protein expression levels were significantly upregulated after treatment with MOC, CI-994 and PAN, i.e. a maximum of 2.1- and 1.3-fold, respectively. Analysis of PAN-treated BON-1 xenografts solely demonstrated increased SSTR2 mRNA expression levels. Comparison of HDACs and SSTR2 expression in BON-1 and NCI-H69 xenografts showed a significantly higher expression of 6/11 HDACs in BON-1 xenografts. Of these HDACs, a significant inverse correlation was found between HDAC3 and SSTR2 expression (Pearson r = −0.92) in the studied cell lines. Significance: To conclude, tumoral uptake levels of radiolabeled DOTA-TATE were not enhanced after HDACi treatment in vivo, but, depending on the applied inhibitor, increased SSTR2 expression levels were observed.</p
Ghrelin drives GH secretion during fasting in man
OBJECTIVES: In humans, fasting leads to elevated serum GH concentrations.
Traditionally, changes in hypothalamic GH-releasing hormone and
somatostatin release are considered as the main mechanisms that induce
this elevated GH secretion during fasting. Ghrelin is an endogenous ligand
of the GH secretagogue receptor and is synthesized in the stomach. As
ghrelin administration in man stimulates GH release, while serum ghrelin
concentrations are elevated during fasting in man, this increase in
ghrelin levels might be another mechanism whereby fasting results in
stimulation of GH release. DESIGN AND SUBJECTS: In ten healthy non-obese
males we performed a double-blind placebo-controlled crossover study
comparing fasting with and fasting without GH receptor blockade. GH,
ghrelin, insulin, glucose and free fatty acids were assessed. RESULTS:
While ghrelin levels do not vary considerably in the fed state, fasting
rapidly induced a diurnal rhythm in ghrelin concentrations. These changes
in serum ghrelin concentrations during fasting were followed by similar,
profound changes in serum GH levels. The rapid development of a diurnal
ghrelin rhythm could not be explained by changes in insulin, glucos
Uptake of thyroxine in cultured anterior pituitary cells of euthyroid rats
The uptake of [125I]T4 was investigated in cultured anterior pituitary
cells isolated from adult fed Wistar rats and cultured for 3 days in
medium containing 10% fetal calf serum. Experiments were performed with
[125I]T4 (10(5) to 2 x 10(6) cpm; 0.35-7 nM) in medium containing 0.5% or
0.1% BSA. The uptake of [125I]T4 increased with time and showed
equilibrium after around 1 h of incubation. The presence of 10 microM
unlabeled T4 during incubation decreased the uptake of [125I]T4 by 65-70%
at all time intervals. After 24 h of incubation, 1.5% iodide and 3.2%
conjugates were detected in the medium, whereas around 20% of cellular
radioactivity represented [125I]T3. The 15-min uptake of [125I]T4 was
significantly reduced by simultaneous incubation with 100 nM T4 (by 24%; P
< 0.05), 100 nM T3 (by 38%; P < 0.001), or 10 microM rT3 (by 32%; P <
0.001), whereas 10 microM tetraiodothyroacetic acid (Tetrac) had no
effect. Furthermore, preincubation (30 min) and incubation (15 min) with
10 microM monodansylcadaverine, oligomycin, or monensin reduced the uptake
of [125I]T4 by 30%, 50%, and 40%, respectively (all P < 0.001).
Substitution of Na+ in the buffer by K+ diminished the uptake of [125I]T4
by 39% (P < 0.005); 2 mM phenylalanine, tyrosine, or tryptophan reduced
[125I]T4 uptake by 18% (P < 0.05), 18% (P = NS), and 33% (P < 0.005),
respectively. Our data suggest that the pituitary contains a specific
carrier-mediated energy-requiring mechanism for [125I]T4 uptake that is
partly dependent on the Na+ gradient. In addition, part of [125I]T4 uptake
in the pituitary might occur through an amino acid transport system. When
expressed per pM of free hormone, the 15-min uptake of [125I]T4 was
approximately as high as that of [125I]T3. Because the reduction of
[125I]T4 uptake by T4, T3, monodansylcadaverine, oligomycin, and monensin
was roughly the same as the previously reported reduction of [125I]T3
uptake by the same compounds, it is further suggested that T4 and T3 share
a common carrier in cultured anterior pituitary cells
Dissociation between the effects of somatostatin (SS) and octapeptide SS-analogs on hormone release in a small subgroup of pituitary- and islet cell tumors
The effects of somatostatin (SS-14 and/or SS-28) and of the three
octapeptide SS-analogs that are available for clinical use (octreotide,
BIM-23014 and RC-160) on hormone release by primary cultures of 15
clinically nonfunctioning pituitary adenomas (NFA), 7 prolactinomas, and 2
insulinomas were investigated. In the pituitary adenoma cultures, a
comparison was made with the effects of the dopamine (DA) agonists
bromocriptine and/or quinagolide. In 5 NFAs, 2 prolactinomas and 1
insulinoma somatostatin receptor (subtype) expression was determined by
ligand binding studies and by in situ hybridization to detect sst1, sst2,
and sst3 messenger RNAs (mRNAs). Four NFA cultures did not secrete
detectable amounts of alpha-subunit, FSH, and/or LH. In the other
cultures, hormone and/or subunit release was inhibited by DA-agonists (10
nM) in 9 of 11, by SS (10 nM) in 7 of 11, and by octapeptide SS-analogs
(10 nM) in 3 of 10 cultures. In three NFA cultures, hormone release was
sensitive to SS but not to SS-analogs. In all cultures, except for one,
DA-agonists were the most effective in inhibiting hormone release. In the
prolactinoma cultures, PRL release was inhibited by DA-agonists (10 nM) in
7 of 7, by SS in 4 of 4, and by octapeptide SS-analogs in 3 of 7 cultures.
A dissociation between the effects of SS and SS-analogs was found in 3
cases. In the cultures sensitive to both bromocriptine and SS-28,
bromocriptine was the most potent compound in 2 out of 4 cultures. In the
2 other cultures, both compounds were equally effective. In 2 insulinoma
cultures, insulin release was inhibited by SS, and by octapeptide
SS-analogs in only one. The presence or absence of an inhibitory effect by
octreotide was in all cases in parallel with the presence or absence of
the inhibitory effect by BIM-23014 and RC-160. Autoradiographic studies
using [125I-Tyr0]SS28 showed specific binding in 4 of 5 NFAs, 1 of 2
prolactinomas, and 1 of 1 insulinoma. Specific [125I-Tyr3]octreotide
binding was found in 2 of 5 NFAs, in 1 of 2 prolactinomas, and in the
insulinoma. Two NFAs showed binding of SS28, but not of the sst2.5
specific ligand octreotide. The tumors showed variable sst1 and/or sst3
mRNA expression, whereas no sst2 expression was found. In conclusion, a
dissociation between the inhibitory effects of SS on the one hand and of
the octapeptide SS-analogs octreotide, BIM-23014 and RC-160 on the other
hand, is observed in a small subgroup of NFAs, prolactinomas, and
insulinomas, suggesting that novel sst subtype specific SS-analogs might
be of benefit in the treatment of selected patients with somatostatin
receptor positive secreting tumors not resp
Radioiodinated somatostatin analogue RC-160: preparation, biological activity, in vivo application in rats and comparison with [123I-Tyr3]octreotide
We have evaluated the potential usefulness of the radioiodinated octapeptide RC-160, a somatostatin analogue, which might serve as a radiopharmaceutical for the in vivo detection of somatostatin receptor-positive tumours. For this purpose, iodine-123 and iodine-125 labelled RC-160 was tested for biological activity and applied in vivo in rats bearing the transplantable rat pancreatic tumour CA20948, which expresses somatostatin receptors. Our group has recently described the in vivo visualization of such tumours in rats and in humans with the radioiodinated somatostatin analogue [Tyr3]octreotide. Like [123I-Tyr3]octreotide, 123I-RC-160 showed uptake in and specific binding in vivo to somatostatin receptor-positive organs and tumours. However, blood radioactivity (background) was higher, resulting in a lower tumour to blood (background) ratio. We therefore conclude that in this animal model 123I-RC-160 has no advantage over [123I-Tyr3]octreotide as a radiopharmaceutical for the in vivo use as a somatostatin receptor imager, although, like [123I-Tyr3]octreotide, 123I-RC-160 shows specific binding to different somatostatin receptor-positive organs. Recently differences were reported in affinity between somatostatin and its analogues for somatostatin receptors expressed in different human cancers, like those of the breast, ovary, exocrine pancreas, prostate and colon. Therefore 123I-RC-160 might be of interest for future use in humans as a radiopharmaceutical for imaging octreotide receptor-negative tumours
Somatostatin analogues for receptor targeted photodynamic therapy
Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2+ AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate
A new radiolabelled somatostatin analogue [111In-DTPA-D-Phe1]RC-160: preparation, biological activity, receptor scintigraphy in rats and comparison with [111In-DTPA-D-Phe1]octreotide
We have evaluated the potential usefulness of indium-111 labelled [DTPA-D-Phe1]RC-160, derived from the octapeptide somatostatin analogue RC-160, as a radiopharmaceutical for the in vivo detection of somatostatin receptor-positive tumours. For this purpose 111In-and 111In-labelled [DTPA-D-Phe1]RC-160 was tested for its biological activity, and applied for somatostatin receptor scintigraphy in vivo to rats bearing the transplantable rat pancreatic tumour CA20948, which expresses somatostatin receptors. We previously described the development of the 111In-labelled somatostatin analogue [DTPA-D-Phe1]octreotide and its use in the in vivo visualization of somatostatin receptor-positive tumours in rats and in humans. Like [111In-DTPA-D-Phe1]octreotide, [111In-DTPA-D-Phe1]RC-160 showed uptake in and specific binding in vivo to somatostatin receptor-positive organs and tumours, and the tumours were clearly visualized by gamma camera scintigraphy. However, as compared to [111In-DTPA-D-Phe1]octreotide, blood radioactivity (background) was higher, resulting in a lower tumour to blood (background) ratio. Using this animal model we therefore conclude that [111In-DTPA-DPhe1]RC-160 has no advantage over [111In-DTPA-DPhe1]octreotide as a radiopharmaceutical in the visualization of somatostatin receptors which bind both analogues. However, recent reports suggest the existence of different somatostatin receptor subtypes on some human cancers, which differentially bind RC-160 and not octreotide. These tumours include cancers of the breast, ovary, exocrine pancreas, prostate and colon. [111In-DTPA-D-Phe1]RC-160 might be of interest for future use in such cancer patients as a radiopharmaceutical for imaging somatostatin receptor-positive tumours, which do not bind octreotide
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